Influenza A virus infection is one of the major causes of human respiratory diseases with an average mortality rate of 36,000/year in the United States alone (CDC, 2007). Respiratory disease caused by Influenza A virus infection can be very severe especially in very young children and the elderly (Fiore et al., 2008, “Prevention and control of influenza: recommendations of the Advisory Committee on Immunization Practices (ACIP),” MMWR Recomm Rep 57(RR-7):1-60). Apart from yearly seasonal outbreaks, IAV can cause frequent epidemics and occasional pandemics in humans (Cox et al., 2005, “Orthomyxoviruses: influenza,” IN Topley and Wilson's Microbiology and Microbial Infections. London: Hodder Arnold Press, pp. 634-98; and Palese & Shaw, 2007, “Orthomyxoviridae: the viruses and their replication,” In Howley, ed., Fields Virology, 5th Edition, Philadelphia, Pa.: Lippincott Williams & Wilkins, pp 1647-1689). The recent emergence of highly pathogenic avian H5N1 virus and the pandemic swine-origin 2009 A (H1N1) influenza virus underscore the threat posed by influenza viruses not only to humans but also to domestic animals and birds (Beigel, J. H., J. Farrar, A. M. Han, F. G. Hayden, R. Hyer, M. D. de Jong, S. Lochindarat, T. K. Nguyen, T. H. Nguyen, T. H. Tran, A. Nicoll, S. Touch, and K. Y. Yuen. 2005. Avian influenza A (H5N1) infection in humans. N Engl J Med 353:1374-85, Korteweg, C., and J. Gu. 2008. Pathology, molecular biology, and pathogenesis of avian influenza A (H5N1) infection in humans. Am J Pathol 172:1155-70, Ong, C. W., K. Y. Ho, L. Y. Hsu, A. Y. Lim, D. A. Fisher, and P. A. Tambyah. 2009. Reacting to the emergence of swine-origin influenza A H1N1. Lancet Infect Dis 9:397-8). Vaccination has been one of the most effective means of protection against influenza virus infection. In addition, there are two categories of FDA approved drugs used for treatment of IAV infections, M2 inhibitors, which block viral uncoating and entry (amantadine and rimantadine) and NA inhibitors, which block viral spreading (oseltamivir and zanamivir; reviewed in von Itzstein, 2007, “The war against influenza: discovery and development of sialidase inhibitors,” Nat Rev Drug Discov 6(12):967-974; De Clercq, 2006, “Antiviral agents active against influenza A viruses,” Nat Rev Drug Discov 5(12):1015-1025; Hayden & Pavia, 2006, “Antiviral management of seasonal and pandemic influenza,” J Infect Dis 194 Suppl 2:S119-126; Sugrue et al., 2008, “Antiviral drugs for the control of pandemic influenza virus,” Ann Acad Med Singapore 37(6):518-524). However, Influenza virus undergoes rapid antigenic evolution through constant genetic reassortment and by accumulating mutations. Consequently, the imperative need for new vaccine strategies and annual vaccination puts an enormous burden on the healthcare system.
Influenza A virus, a member of the Orthomyxovirus family, is an enveloped virus. Its genome consists of eight single-stranded, negative sense RNA segments (PB1, PB2, PA, HA, NP, NA, M and NS) (Palese, P., and M. L. Shaw. 2007. Orthomyxoviridae: the viruses and their replication, p. 1647-1689. In D. M. K. P. M. Howley (ed.), Fields virology, 5th Edition ed. Lippincott Williams & Wilkins, Philadelphia, Pa.). During infection 10 or 11 viral proteins are expressed in the cells. The M and NS segments express two proteins each from alternatively spliced mRNAs (Gibbs, J. S., D. Malide, F. Hornung, J. R. Bennink, and J. W. Yewdell. 2003.) The influenza A virus PB1-F2 protein targets the inner mitochondrial membrane via a predicted basic amphipathic helix that disrupts mitochondrial function. J Virol 77:7214-24). NS1 and NEP are made from the NS segment, the smallest segment in influenza virus. NS1 is a multifunctional protein that counteracts host antiviral response by blocking numerous pathways (Hale, B. G., R. E. Randall, J. Ortin, and D. Jackson. 2008. The multifunctional NS1 protein of influenza A viruses. J Gen Virol 89:2359-76). NEP is involved in the export of vRNA from the nucleus (O'Neill, R. E., J. Talon, and P. Palese. 1998. The influenza virus NEP (NS2 protein) mediates the nuclear export of viral ribonucleoproteins. EMBO J 17:288-96). Development of a reverse genetics system for influenza viruses has tremendously helped in the identification of several viral genetic factors that contribute to severe pathogenicity (Fodor, E., L. Devenish, O. G. Engelhardt, P. Palese, G. G. Brownlee, and A. Garcia-Sastre. 1999. Rescue of influenza A virus from recombinant DNA. J Virol 73:9679-82, Neumann, G., T. Watanabe, H. Ito, S. Watanabe, H. Goto, P. Gao, M. Hughes, D. R. Perez, R. Donis, E. Hoffmann, G. Hobom, and Y. Kawaoka. 1999. Generation of influenza A viruses entirely from cloned cDNAs. Proc Natl Acad Sci USA 96:9345-50), as well as in the study different aspects of the virus life cycle. The reverse genetics system can also be used for faster generation of pandemic vaccines and development influenza based vaccine (Gao, Q., E. W. Brydon, and P. Palese. 2008. A seven-segmented influenza A virus expressing the influenza C virus glycoprotein HEF. J Virol 82:6419-26, Koudstaal, W., L. Hartgroves, M. Havenga, I. Legastelois, C. Ophorst, M. Sieuwerts, D. Zuijdgeest, R. Vogels, J. Custers, E. de Boer-Luijtze, O. de Leeuw, L. Cornelissen, J. Goudsmit, and W. Barclay. 2009. Suitability of PER.C6 cells to generate epidemic and pandemic influenza vaccine strains by reverse genetics. Vaccine 27:2588-93, Steel, J., S. V. Burmakina, C. Thomas, E. Spackman, A. Garcia-Sastre, D. E. Swayne, and P. Palese. 2008. A combination in-ovo vaccine for avian influenza virus and Newcastle disease virus. Vaccine 26:522-31).
Influenza viruses use sialic acid as cellular receptors to enter target cells (Palese, P., and M. L. Shaw. 2007. Orthomyxoviridae: the viruses and their replication, p. 1647-1689. In D. M. K. P. M. Howley (ed.), Fields virology, 5th Edition ed. Lippincott Williams & Wilkins, Philadelphia, Pa.). Several studies have shown that influenza virus can infect a variety of cell types, both in vitro and in vivo (Hao, X., T. S. Kim, and T. J. Braciale. 2008. Differential response of respiratory dendritic cell subsets to influenza virus infection. J Virol 82:4908-19, Kim, T. S., and T. J. Braciale. 2009. Respiratory dendritic cell subsets differ in their capacity to support the induction of virus-specific cytotoxic CD8+ T cell responses. PLoS One 4:e4204, Kumlin, U., S. Olofsson, K. Dimock, and N. Arnberg. 2008. Sialic acid tissue distribution and influenza virus tropism. Influenza Other Respi Viruses 2:147-54).
Although studies of influenza A virus using animal models and tissue culture have provided tremendous knowledge about both the virus and host factors which determine pathogenesis, following viral infection and pathogenesis in vivo may provide us with a better picture of the complex interactions between the virus and the host (8). Such in vivo studies have been hampered primarily due to the lack of fully competent reporter viruses (Knipe, D. M. 2007. Field's Virology 5th Ed; Orthomyxoviruses, Neumann, G., and Y. Kawaoka. 2006. Host range restriction and pathogenicity in the context of influenza pandemic. Emerg Infect Dis 12:881-6).